Christian R. H. Raetz

Lipidomics reveals a remarkable diversity of lipids in human plasma

The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patients blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

Bacterial polysaccharide synthesis and gene nomenclature

Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.

LMSD: LIPID MAPS structure database

The LIPID MAPS Structure Database (LMSD) is a relational database encompassing structures and annotations of biologically relevant lipids. Structures of lipids in the database come from four sources: (i) LIPID MAPS Consortiums core laboratories and partners; (ii) lipids identified by LIPID MAPS experiments; (iii) computationally generated structures for appropriate lipid classes; (iv) biologically relevant lipids manually curated from LIPID BANK, LIPIDAT and other public sources. All the lipid structures in LMSD are drawn in a consistent fashion. In addition to a classification-based retrieval of lipids, users can search LMSD using either text-based or structure-based search options. The text-based search implementation supports data retrieval by any combination of these data fields: LIPID MAPS ID, systematic or common name, mass, formula, category, main class, and subclass data fields. The structure-based search, in conjunction with optional data fields, provides the capability to perform a substructure search or exact match for the structure drawn by the user. Search results, in addition to structure and annotations, also include relevant links to external databases. The LMSD is publicly available at

A left-handed parallel beta helix in the structure of UDP-N-acetylglucosamine acyltransferase.

UDP-N-acetylglucosamine 3-O-acyltransferase (LpxA) catalyzes the transfer of (R)-3-hydroxymyristic acid from its acyl carrier protein thioester to UDP-N-acetylglucosamine. LpxA is the first enzyme in the lipid A biosynthetic pathway and is a target for the design of antibiotics. The x-ray crystal structure of LpxA has been determined to 2.6 angstrom resolution and reveals a domain motif composed of parallel β strands, termed a left-handed parallel β helix (LβH). This unusual fold displays repeated violations of the protein folding constraint requiring right-handed crossover connections between strands of parallel β sheets and may be present in other enzymes that share amino acid sequence homology to the repeated hexapeptide motif of LpxA.

Function of Escherichia coli MsbA, an Essential ABC Family Transporter, in Lipid A and Phospholipid Biosynthesis

The Escherichia coli msbA gene, first identified as a multicopy suppressor of htrB mutations, has been proposed to transport nascent core-lipid A molecules across the inner membrane (Polissi, A., and Georgopoulos, C. (1996) Mol. Microbiol. 20, 1221–1233). msbA is an essentialE. coli gene with high sequence similarity to mammalian Mdr proteins and certain types of bacterial ABC transporters.htrB is required for growth above 32 °C and encodes the lauroyltransferase that acts after Kdo addition during lipid A biosynthesis (Clementz, T., Bednarski, J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095–12102). By using a quantitative new 32Pi labeling technique, we demonstrate that hexa-acylated species of lipid A predominate in the outer membranes of wild type E. coli labeled for several generations at 42 °C. In contrast, in htrB mutants shifted to 42 °C for 3 h, tetra-acylated lipid A species and glycerophospholipids accumulate in the inner membrane. Extra copies of the cloned msbA gene restore the ability ofhtrB mutants to grow at 42 °C, but they do not increase the extent of lipid A acylation. However, a significant fraction of the tetra-acylated lipid A species that accumulate in htrBmutants are transported to the outer membrane in the presence of extra copies of msbA. E. coli strains in which msbA synthesis is selectively shut off at 42 °C accumulate hexa-acylated lipid A and glycerophospholipids in their inner membranes. Our results support the view that MsbA plays a role in lipid A and possibly glycerophospholipid transport. The tetra-acylated lipid A precursors that accumulate in htrB mutants may not be transported as efficiently by MsbA as are penta- or hexa-acylated lipid A species.

Regulated Accumulation of Desmosterol Integrates Macrophage Lipid Metabolism and Inflammatory Responses

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

Solution structure and dynamics of the outer membrane enzyme PagP by NMR

The bacterial outer membrane enzyme PagP transfers a palmitate chain from a phospholipid to lipid A. In a number of pathogenic Gram-negative bacteria, PagP confers resistance to certain cationic antimicrobial peptides produced during the host innate immune response. The global fold of Escherichia coli PagP was determined in both dodecylphosphocholine and n-octyl-β-d-glucoside detergent micelles using solution NMR spectroscopy. PagP consists of an eight-stranded anti-parallel β-barrel preceded by an amphipathic α helix. The β-barrel is well defined, whereas NMR relaxation measurements reveal considerable mobility in the loops connecting individual β-strands. Three amino acid residues critical for enzymatic activity localize to extracellular loops near the membrane interface, positioning them optimally to interact with the polar headgroups of lipid A. Hence, the active site of PagP is situated on the outer surface of the outer membrane. Because the phospholipids that donate palmitate in the enzymatic reaction are normally found only in the inner leaflet of the outer membrane, PagP activity may depend on the aberrant migration of phospholipids into the outer leaflet. This finding is consistent with an emerging paradigm for outer membrane enzymes in providing an adaptive response toward disturbances in the outer membrane.

Transfer of palmitate from phospholipids to lipid A in outer membranes of Gram-negative bacteria

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram‐negative bacteria. The Salmonella typhimurium PhoP/PhoQ‐activated gene pagP is required both for biosynthesis of hepta‐acylated lipid A species containing palmitate and for resistance to cationic anti‐microbial peptides. Palmitoylated lipid A can also function as an endotoxin antagonist. We now show that pagP and its Escherichia coli homolog (crcA) encode an unusual enzyme of lipid A biosynthesis localized in the outer membrane. PagP transfers a palmitate residue from the sn‐1 position of a phospholipid to the N‐linked hydroxymyristate on the proximal unit of lipid A (or its precursors). PagP bearing a C‐terminal His6‐tag accumulated in outer membranes during overproduction, was purified with full activity and was shown by cross‐linking to behave as a homodimer. PagP is the first example of an outer membrane enzyme involved in lipid A biosynthesis. Additional pagP homologs are encoded in the genomes of Yersinia and Bordetella species. PagP may provide an adaptive response toward both Mg2+ limitation and host innate immune defenses.

An Inner Membrane Enzyme in Salmonellaand Escherichia coli That Transfers 4-Amino-4-deoxy-l-arabinose to Lipid A INDUCTION IN POLYMYXIN-RESISTANT MUTANTS AND ROLE OF A NOVEL LIPID-LINKED DONOR

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance inEscherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or twol-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on theS. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, oryfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IVA, a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesizel-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132–43144), ArnT utilizes the novel lipid undecaprenyl phosphate-α-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.

An Escherichia coli Mutant Defective in Lipid Export

Escherichia coli phospholipids and lipopolysaccharide, made on the inner surface of the inner membrane, are rapidly transported to the outer membrane by mechanisms that are not well characterized. We now report a temperature-sensitive mutant (WD2) with an A270T substitution in a trans-membrane region of the ABC transporter MsbA. As shown by32Pi and 14C-acetate labeling, export of all major lipids to the outer membrane is inhibited by ∼90% in WD2 after 30 min at 44 °C. Transport of newly synthesized proteins is not impaired. Electron microscopy shows reduplicated inner membranes in WD2 at 44 °C, consistent with a key role for MsbA in lipid trafficking.